Metabolism, Disposition, Excretion, and Potential Transporter Inhibition of 7-16, an Improving 5-HT2A Receptor Antagonist and Inverse Agonist for Parkinson's Disease.

Compound 7-16 was designed and synthesized in our previous study and was identified as a more potential selective 5-HT2A receptor antagonist and inverse agonist for treating Parkinson's disease psychosis (PDP). Then, the metabolism, disposition, and excretion properties of 7-16 and its potential inhibition on transporters were investigated in this study to highlight advancements in the understanding of its therapeutic mechanisms. The results indicate that a total of 10 metabolites of 7-16/[14C]7-16 were identified and determined in five species of liver microsomes and in rats using UPLC-Q Exactive high-resolution mass spectrometry combined with radioanalysis. Metabolites formed in human liver microsomes could be covered by animal species. 7-16 is mainly metabolized through mono-oxidation (M470-2) and N-demethylation (M440), and the CYP3A4 isozyme was responsible for both metabolic reactions. Based on the excretion data in bile and urine, the absorption rate of 7-16 was at least 74.7%. 7-16 had weak inhibition on P-glycoprotein and no effect on the transport activity of OATP1B1, OATP1B3, OAT1, OAT3, and OCT2 transporters. The comprehensive pharmacokinetic properties indicate that 7-16 deserves further development as a new treatment drug for PDP.

Efficacy of Temozolomide-Conjugated Gold Nanoparticle Photothermal Therapy of Drug-Resistant Glioblastoma and Its Mechanism Study.

Temozolomide (TMZ) is a standard-of-care chemotherapeutic drug for the treatment of glioblastoma (GBM), but TMZ-acquired resistance limits its therapeutic effect. In this study, TMZ-loaded gold nanoparticles (TMZ@GNPs) with anti-EphA3 modification on the surface (anti-EphA3-TMZ@GNPs) were synthesized for chemical and auxiliary plasma photothermal treatment (GNPs-PPTT), aiming to overcome the problem of glioma resistance to TMZ and improve the therapeutic effects of GBM. The prepared anti-EphA3-TMZ@GNPs were spherical with a particle size of 45.88 ± 1.9 nm, and the drug loading was 7.31 ± 0.38%. In vitro, cell-culture-based experiments showed that anti-EphA3 increased the cellular uptake of GNPs in T98G cells. Upon laser irradiation, the cytotoxicity and apoptosis rate in the anti-EphA3-TMZ@GNPs-treated group were significantly higher than those in the GNPs and nonphotothermal groups (p < 0.001). The Western blot analysis showed that the GNPs-PPTT-mediated killing of tumor cells induced apoptosis by regulating the apoptotic signaling molecules and cell cycle inhibitors; the expression of MGMT significantly decreased upon p53 induction, thereby reversing drug resistance. After photothermal treatment, the survival time of the subcutaneous GBM model of nude mice in the anti-EphA3-TMZ@GNPs group was prolonged to 46 days, 1.64-fold longer as compared to that in the TMZ group. Based on H&E and TUNEL staining, GNPs-PPTT could elevate apoptosis in T98G cells. In vivo thermal imaging results showed that GNPs could enter the brain via intranasal administration and be eliminated in 2 days, indicating that GNPs are safe for brain. In conclusion, GNPs-PPTT could effectively induce apoptosis in glioma cells and reverse TMZ resistance, thereby, indicative of a promising treatment strategy for GBM.

Intranasal Delivery of Temozolomide-Conjugated Gold Nanoparticles Functionalized with Anti-EphA3 for Glioblastoma Targeting.

Glioblastoma multiforme (GBM) is a highly lethal and aggressive tumor of the brain that carries a poor prognosis. Temozolomide (TMZ) has been widely used as a first-line treatment for GBM. However, poor brain targeting, side effects, and drug resistance limit its application for the treatment of GBM. We designed a Temozolomide-conjugated gold nanoparticle functionalized with an antibody against the ephrin type-A receptor 3 (anti-EphA3-TMZ@GNPs) for targeted GBM therapy via intranasal administration. The system can bypass the blood-brain barrier and target active glioma cells to improve the glioma targeting of TMZ and enhance the treatment efficacy, while reducing the peripheral toxicity and drug resistance. The prepared anti-EphA3-TMZ@GNPs were 46.12 ± 2.0 nm and suitable for intranasal administration, which demonstrated high safety to the nasal mucosa in a toxicity assay. In vitro studies showed that anti-EphA3-TMZ@GNPs exhibited significantly enhanced cellular uptake and toxicity, and a higher cell apoptosis ratio has been seen compared with that of TMZ (54.9 and 14.1%, respectively) toward glioma cells (C6). The results from experiments on TMZ-resistant glioma cells (T98G) demonstrated that the IC50 of anti-EphA3-TMZ@GNPs (64.06 ± 0.16 µM) was 18.5-fold lower than that of TMZ. In addition, Western blot analysis also revealed that anti-EphA3-TMZ@GNPs effectively down-modulated expression of O6-methylguanine-DNA methyltransferase and increased chemosensitivity of T98G to TMZ. The antiglioma efficacy in vivo was investigated in orthotopic glioma-bearing rats, and the results demonstrated that the anti-EphA3-TMZ@GNPs prolonged the median survival time to 42 days and increased tumor-cell apoptosis dramatically compared with TMZ. In conclusion, anti-EphA3-TMZ@GNPs could serve as an intranasal drug delivery system for efficacious treatment of GBM.

A novel LC-MS/MS approach to the pharmacokinetic study of free and bound aflibercept simultaneously.

To comprehensively evaluate the pharmacokinetic (PK) characteristics of aflibercept, we established a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method to determine the concentration of vascular endothelial growth factor (VEGF)-A-bound aflibercept and free aflibercept. A specific sample preparation method of nano-surface and molecular-orientation limited (nSMOL) proteolysis was performed to extract both free and bound aflibercept from plasma. The tryptic peptides unique to aflibercept and VEGF-A were selected to quantify the amounts of total aflibercept and aflibercept-VEGF complex, respectively. The method was validated by evaluating its selectivity, linearity, precision, accuracy, extraction recovery, matrix effect, and stability. It was then successfully used to quantify total and bound aflibercept concentrations in cynomolgus monkey plasma, while indirectly obtaining the concentration of free aflibercept by subtraction. The PK results of this LC-MS/MS method are comparable to the traditional enzyme-linked immunosorbent assay (ELISA) results. It is thus a reliable and complementary method for the PK evaluation of aflibercept. Graphical abstract.

Effect of palmitic acid on the characteristics and release profiles of rotigotine-loaded microspheres.

BACKGROUND: The initial burst release is a major obstacle to the development of microsphere-formulated drug products. PURPOSE: To investigate the influence of palmitic acid on the characteristics and release profiles of rotigotine-loaded poly(d,l-lactide-co-glycolide) microspheres. MATERIALS AND METHODS: Rotigotine-loaded microspheres (RMS) were prepared using the oil-in-water emulsion solvent evaporation technique. The in vitro characteristics of the RMS were evaluated with scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and a particle size analyzer. The in vitro drug release and in vivo pharmacokinetics of the RMS were investigated. RESULTS AND DISCUSSION: The SEM results showed that the addition of palmitic acid changed the surface morphology of the microspheres from smooth to dimpled and then to non-smooth as the palmitic acid content increased. DSC revealed the existence of molecularly dispersed forms of palmitic acid in the microspheres. The in vitro and in vivo release profiles indicated that the addition of 5% and 8% palmitic acid significantly decreased the burst release of rotigotine from the microspheres, and the late-stage release was delayed as the palmitic acid content increased across the investigated range (5-15%). CONCLUSION: The addition of palmitic acid to the microspheres significantly affects the release profile of rotigotine from RMS.

Development, validation and application of an ion-pair reversed-phase liquid chromatography-tandem mass spectrometry method for the quantification of nusinersen.

Background: The fully phosphorothioate-modified oligonucleotide (OGN) nusinersen has low ionization efficiency in the negative ion mode, resulting in a low mass spectrometry response. There have been no relevant reports on developing a LC-MS method for the determination of nusinersen by optimizing mobile phase composition. Materials & methods: Mobile phase additives comprised of 15 mM triethylamine/25 mM 1,1,1,3,3,3-hexafluoro-2-propanol with a pH of 9.6. Nusinersen was extracted from plasma using Oasis® HLB solid-phase extraction (Waters, MA, USA). Results & conclusion: By adjusting the pH of the mobile phase to 9.6 by optimizing the type and concentration of ion-pair reagents, a high mass spectrometry response was obtained. The developed method was applied to nusinersen and met the requirements for the pharmacokinetic study of nusinersen in rabbits.

Development and validation of an HILIC/MS/MS method for determination of nusinersen in rabbit plasma.

A hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC/MS/MS) method was developed and validated for the quantitative analysis of the fully phosphorothioate modified oligonucleotide nusinersen. HILIC/MS/MS method is more robust and compatible with mass spectrometry than ion pair reversed-phase liquid chromatography-tandem mass spectrometry (IP-RP-LC/MS/MS). Various types and concentrations of additives and different pH of mobile phase affected the mass spectrometry response, chromatographic peak shape and retention of nusinersen. The optimized extraction method of nusinersen employs hydrophilic-lipophilic balance solid phase extraction, with a recovery of up to 80 %. Chromatographic quantification was performed using a gradient system on an amide column and the mobile phase consisted of ammonium acetate, acetonitrile and water in a certain proportion. The fully phosphorothioate modified nusinersen can obtain a high mass spectrometry response by providing greater peak symmetry and high ionization efficiency in a high-pH mobile phase. Moreover, the significant carry over interference was observed at the pH 6.3 of the mobile phase. Adjusting the pH value up to 10, and the carry over interference disappeared. The lower limit of quantitation of this developed HILIC/MS/MS assay was 30.0 ng/mL and the method was systematic methodology validated. This HILIC/MS/MS method provides an attractive and robust alternative for the quantitative analysis of nusinersen and was applied in the pharmacokinetic study of nusinersen in rabbits.

A novel scaffold long-acting selective estrogen receptor antagonist and degrader with superior preclinical profile against ER+ breast cancer.

Breast cancer is one of the most common malignant tumors in women worldwide, with the majority of cases showing expression of estrogen receptors (ERs). Although drugs targeting ER have significantly improved survival rates in ER-positive patients, drug resistance remains an unmet clinical need. Fulvestrant, which overcomes selective estrogen receptor modulator (SERM) and AI (aromatase inhibitor) resistance, is currently the only long-acting selective estrogen receptor degrader (SERD) approved for both first and second-line settings. However, it fails to achieve satisfactory efficacy due to its poor solubility. Therefore, we designed and synthesized a series of novel scaffold (THC) derivatives, identifying their activities as ER antagonists and degraders. G-5b, the optimal compound, exhibited binding, antagonistic, degradation or anti-proliferative activities comparable to fulvestrant in ER+ wild type and mutants breast cancer cells. Notably, G-5b showed considerably improved stability and solubility. Research into the underlying mechanism indicated that G-5b engaged the proteasome pathway to degrade ER, subsequently inhibiting the ER signaling pathway and leading to the induction of apoptosis and cell cycle arrest events. Furthermore, G-5b displayed superior in vivo pharmacokinetics and pharmacodynamics properties, coupled with a favorable safety profile in the MCF-7 tamoxifen-resistant (MCF-7/TR) tumor xenograft model. Collectively, G-5b has emerged as a highly promising lead compound, offering potent antagonistic and degradation activities, positioning it as a novel long-acting SERD worthy of further refinement and optimization.

Optimization and validation of the liquid chromatography coupled to tandem mass spectrometry method for assessing octreotide release from microspheres during inflammation in rabbit models.

To study the effect of acute-phase reaction (APR) of inflammation on the release of octreotide acetate microsphere (Sandostatin®, SLAR) at a clinical dose, a more sensitive liquid chromatography coupled to tandem mass spectrometry analysis method needs to be developed because of the low plasma concentrations of octreotide. Solid-phase microextraction with an Oasis® HLB µElution plate was adopted for sample preparation. Extraction recovery ranged from 65.7 % to 73.2 %, and the matrix effect was negligible. High sensitivity and an intense chromatographic peak were acquired by optimizing the chromatography and mass spectrometry conditions. The lower limit of quantitation (LLOQ) was 0.01 ng/mL based on 100 µL of plasma, and linearity ranged from 0.01 to 5.0 ng/mL. The coefficients of variations for intraday and interday precision were less than 4.4 %, and the relative error of accuracy was within 5.7 %. The validated method was successfully applied to pharmacokinetics studies of SLAR in a seven-day inflammation model of rabbits, indicating that the APR did not affected the release and pharmacokinetics of the octreotide microspheres.

Preparation of rotigotine-loaded microspheres and their combination use with L-DOPA to modify dyskinesias in 6-OHDA-lesioned rats.

PURPOSE: To prepare rotigotine loaded microspheres (RoMS) to achieve continuous dopaminergic stimulation (CDS) for the treatment of Parkinson's disease (PD) and investigate both the therapeutic benefit and inducibility of AIMs of administration of RoMS combination with L-DOPA in 6-OHDA-leisioned rats. METHODS: Rotigotine was encapsulated into poly(lactic-co-glycolic acid) (PLGA) microspheres by an oil-in-water emulsion solvent evaporation technique. In vitro characteristics and in vivo pharmacokinetics of RoMS either in rat blood or brain (by microdialysis) were investigated. Contraversive rotations and AIMs were observed to investigate the therapeutic benefit and the propensity to induce dyskinesia of RoMS or RoMS combination with L-DOPA in 6-OHDA-lesioned rats. RESULTS: RoMS displayed continuous-release characteristics of rotigotine in animals and exhibited a steady efficacy lasted for 2 weeks in 6-OHDA-lesioned rats. No significant difference of the therapeutic benefit between the treatment of RoMS and pulsatile L-DOPA combination and mono L-DOPA was found. While the dyskinesia was significantly decreased with the treatment of RoMS and pulsatile L-DOPA combination compared to mono L-DOPA. CONCLUSIONS: RoMS could supply an alternative of CDS for the treatment of PD and the study indicates a potential advantage of RoMS for the treatment of mild and advanced PD patient in combination with L-DOPA.

Temozolomide hexadecyl ester targeted plga nanoparticles for drug-resistant glioblastoma therapy via intranasal administration.

Introduction: Temozolomide (TMZ) is the first-line drug for glioblastoma (GBM), but it is limited in clinical use due to the drug resistance, poor brain targeting, and side effects. Temozolomide hexadecyl ester (TMZ16e), a TMZ derivative with high lipophilicity, membrane permeability, and high anti-glioma properties, has the potential to reverse drug resistance. In this study, anti-ephrin type-A receptor 3 (EphA3) modified TMZ16e loaded nanoparticles (NPs) were prepared for targeted GBM therapy via intranasal administration to deliver TMZ16e to the brain, treat drug-resistant glioma effectively, and reduce peripheral toxicity. Methods: TMZ16e loaded NPs were prepared by emulsion solvent evaporation method followed by modified with anti-EphA3 (anti-EphA3-TMZ16e-NPs). In vitro evaluations were performed by an MTT assay and flow cytometry analysis. The orthotopic nude mice models were used to evaluate the anti-glioma effect in vivo. Additionally, we investigated the anti-drug resistant mechanism by western blot analysis. Results: The particle size of the prepared NPs was less than 200 nm, and the zeta potential of TMZ16e-NPs and anti-EphA3-TMZ16e-NPs were -23.05 ± 1.48 mV and -28.65 ± 1.20mV, respectively, which is suitable for nasal delivery. In vitro studies have shown that anti-EphA3 modification increased the cellular uptake of nanoparticles in T98G cells. The cytotoxicity in the anti-EphA3-TMZ16e-NPs treated group was significantly higher than that of the TMZ16e-NPs, TMZ16e, and TMZ groups (p < 0.01), and the cell cycle was blocked. Western blotting analysis showed that the TMZ16e-loaded NPs were able to effectively downregulate the expression level of O6-methylguanine-deoxyribonucleic acid-methyltransferase (MGMT) protein in T98G cells and reverse drug resistance. In vivo studies showed that the median survival time of tumor-bearing nude mice in the anti-EphA3-TMZ16e-NPs group was extended to 41 days, which was 1.71-fold higher than that of the saline group and the TUNEL staining results of the brain tissue section indicated that the TMZ16e-loaded NPs could elevate apoptosis in T98G cells. Conclusion: In conclusion, the TMZ16e-loaded NPs can be effectively delivered to the brain and targeted to gliomas, exhibiting better anti-glioma activity, indicating they possess great potential in the treatment of drug-resistant glioma.

Analytical comparability assessment on glycosylation of ziv-aflibercept and the biosimilar candidate.

Ziv-aflibercept (aflibercept) is a recombinant fusion protein which combines the portions of human vascular endothelial growth factor receptors extracellular domains fused to the Fc portion of human IgG1. It is a highly sialylated glycoprotein with 5 N-glycosylation sites. In this study, a comprehensive strategy for comparability study of the complex glycosylation was developed between aflibercept and the biosimilar candidate including the investigations on N-glycosylation sites, site occupancy, site-specific glycoforms, released glycans and sialic acids. The results indicated that same N-glycosylation sites were identified, site occupancy were 100% except N68 site, site-specific glycoforms and released glycans showed similar glycan species, contents of NANA were at a same level for two products. Minor differences were found between two products. The biosimilar candidate presented lower level of aglycosylation, lower level of glycans containing one terminal sialic acid, higher level of glycans containing two terminal sialic acids, higher level of G0F and Man5, lower level of G1F and G2F compared with aflibercept. However, further studies exhibited no differences were observed in the cell-based biological potency and Fc effector function. Moreover, the biosimilar candidate showed a similar pharmacokinetics curve and bioequivalence compared with aflibercept.

Quality markers based on phytochemical analysis and anti-inflammatory screening: An integrated strategy for the quality control of Dalbergia odorifera by UHPLC-Q-Orbitrap HRMS.

BACKGROUND: Quality control, key for the clinical application of traditional Chinese medicines (TCMs), should be connected to the authentication and efficacy of TCMs. The heartwood of Dalbergia odorifera has been widely used to treat inflammation-related diseases. However, in the Chinese pharmacopeia, only the total volatile oil, which does not sufficiently reflect the clinical efficacy, is used as a quality control indicator. PURPOSE: Establishing a "phytochemical-specificity-effectiveness-Q-marker" analytical strategy to improve the quality control of D. odorifera. METHODS: Combined with biosynthetic pathway analysis, phytochemical compositions identified by UHPLC-Q-Orbitrap HRMS were used to build substantial phytochemical groups and further discover specific Q-markers. Then, lipopolysaccharide-stimulated RAW 264.7 cells were used to screen effective anti-inflammatory ingredients. Finally, a UHPLC-HRMS method was developed and validated to quantify the selected Q-markers in D. odorifera samples. RESULTS: Along the constructed biosynthetic pathways, 93 phytochemical components were identified in D. odorifera, including 7 chalcones, 13 flavanones, 21 isoflavones, 21 isoflavanones, 3 flavonols, 19 neoflavones, etc. Among them, 31 compounds representing these 6 categories were further evaluated for their anti-inflammatory activities. It revealed that the extract of D. odorifera and nine flavonoids in the noncytotoxic range could alleviated lipopolysaccharide-stimulated inflammation in RAW 264.7 cells by decreasing the production of proinflammatory mediators such as nitric oxide and interleukin-6. Notably, neoflavones, as species-specific components, exhibited superior anti-inflammatory activities among the representative compounds. Finally, 12 Q-markers (butin, liquiritigenin, eriodictyol, melanettin, naringenin, butein, genistein, 4'-hydroxy-4-methoxydalbergione, isoliquiritigenin, 2,4-dihydroxy-5-methoxybenzophenone, medicarpin, and pinocembrin), which reflect specificity and effectiveness, were successfully quantified in 10 batches of samples from different origins. The origins and consistency of D. odorifera could be efficiently discriminated by hierarchical cluster analysis (HCA). CONCLUSION: The analysis strategy that combines phytochemical analysis with anti-inflammatory screening clarified the therapeutic material basis and discovered Q-markers, which possibly offers a more comprehensive quality assessment of D. odorifera.

Synthesis and Characterization of a Series of Temozolomide Esters and Its Anti-glioma Study.

Temozolomide is a first-line therapeutic drug for glioblastoma (GBM), and it has a low solubility, short biological half-life, and resistance to drug limits in clinical applications. Therefore, it is necessary to find more effective anti-tumor drugs to overcome drug resistance and enhance its anti-glioma activity. We therefore used n-butanol, n-hexanol, n-octanol, 1-dodecanol and 1-hexadecanol to synthesize a series of temozolomide ester compounds (TMZEs) and then investigated their physicochemical properties and anti-glioma efficacy. Our results showed that TMZEs had a higher lipophilicity compared to TMZ and could stably exist in plasma and brain homogenates. TMZEs had significantly increased cytotoxicity and cellular uptake in C6 glioma cells as chain lengths increased. Additionally, the IC50 of TMZ-16E towards TMZ-resistant cells (T98G) was 85.9-fold lower than that of TMZ (p < 0.001), and Western blot results demonstrated that TMZ-16E could significantly reduce the expression of O6-methylguanine-DNA-methyltransferase (MGMT). The in vivo anti-glioma efficacy of TMZ-16E were then investigated in orthotopic and subcutaneous GBM models. TMZ-16E prolonged the survival time to 35 days in orthotopic glioma bearing rats, which was 1.94-fold longer than the survival time of rats treated with TMZ, and TMZ-16E increased tumor cell apoptosis based on TUNEL staining. Moreover, TMZ-16E (50 mg/kg) noticeably slowed the growth of T98G subcutaneous tumors by down-modulating MGMT expression in subcutaneous GBM-bearing mice, indicating that TMZ-16E could effectively reverse drug resistance. In conclusion, TMZEs improved the lipophilicity and stability of these drugs. Especially, TMZ-16E could reverse drug resistance and improve therapeutic effects of TMZ, which has clinical application potential for GBM treatment.

Pharmacological Characterization of Toludesvenlafaxine as a Triple Reuptake Inhibitor.

Toludesvenlafaxine hydrochloride dihydrate is a novel chemical entity and a potential triple monoamine reuptake inhibitor. This study characterized the in vitro triple reuptake inhibition activity, antidepressant-like activity in animals, and pharmacokinetic profiles in rats of toludesvenlafaxine. Binding affinity was determined using human serotonin transporter (SERT) protein, norepinephrine transporter (NET) protein and dopamine transporter (DAT) protein, and the reuptake inhibition was determined using Chinese hamster ovary cells expressing human SERT, NET and DAT. The antidepressant-like activity was examined in rat chronic unpredictable mild stress model and olfactory bulbectomized model. In rats, the tissue distribution and pharmacokinetic parameters were determined. Toludesvenlafaxine had high binding affinity on SERT, NET and DAT, and significantly inhibited the reuptake of serotonin (IC50 = 31.4 ± 0.4 nM), norepinephrine (IC50 = 586.7 ± 83.6 nM) and dopamine (IC50 = 733.2 ± 10.3 nM) in vitro. Toludesvenlafaxine demonstrated significant antidepressant-like effects in rat models at 8-16 mg/kg. In addition, toludesvenlafaxine significantly reduced serum corticosterone and significantly increased testosterone levels in rats. Toludesvenlafaxine was quickly absorbed and converted to O-desvenlafaxine (ODV) after oral administration, both of which were selectively distributed into the hypothalamus with high concentration. Plasma ODV exposure was proportionally related to the doses after oral dosing. These results suggest that toludesvenlafaxine is a triple reuptake inhibitor with relatively fast-acting antidepressant-like activity and good therapeutic profile including improvement of anhedonia and sexual function.

Two novel human anti-CD25 antibodies with antitumor activity inversely related to their affinity and in vitro activity.

High tumor regulatory T (Treg) cell infiltration is associated with poor prognosis of many cancers. CD25 is highly expressed on tumor Treg cells and is a potential target for Treg deletion. Previously characterized anti-CD25 antibodies appear to have limited efficacy in tumor inhibition. Here we identified two human anti-CD25 antibodies, BA9 and BT942, which did not prevent the activation of IL-2R signaling pathway by IL-2. BT942 had weaker binding and cytotoxic activity to human CD25-expressing cell lines than BA9. But both demonstrated significant tumor growth inhibition in early and late-stage animal cancer models. BT942 resulted in a higher expansion of CD8+ T cells and CD4+ T cells in tumor microenvironment in mouse MC38 model compared to BA9. BT942 also demonstrated significant higher tumor growth inhibition and higher expansion of CD8+ T cells and CD4+ T cells in combination with an anti-PD1 antibody. Pharmacokinetic study of BT942 in cynomolgus monkeys demonstrated a half-life of 206.97 ± 19.03 h. Structural analysis by cryo-EM revealed that BT942 recognizes an epitope on opposite side of the CD25-IL-2 binding site, consistent with no IL-2 signaling blockade in vitro. BT942 appears to be an excellent candidate for cancer immunotherapy.

Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing COVID-19 pandemic, which has resulted in more than two million deaths at 2021 February . There is currently no approved therapeutics for treating COVID-19. The SARS-CoV-2 Spike protein is considered a key therapeutic target by many researchers. Here we describe the identification of several monoclonal antibodies that target SARS-CoV-2 Spike protein. One human antibody, CA521FALA, demonstrated neutralization potential by immunizing human antibody transgenic mice. CA521FALA showed potent SARS-CoV-2-specific neutralization activity against SARS-CoV-2 pseudovirus and authentic SARS-CoV-2 infection in vitro. CA521FALA also demonstrated having a long half-life of 9.5 days in mice and 9.3 days in rhesus monkeys. CA521FALA inhibited SARS-CoV-2 infection in SARS-CoV-2 susceptible mice at a therapeutic setting with virus titer of the lung reduced by 4.5 logs. Structural analysis by cryo-EM revealed that CA521FALA recognizes an epitope overlapping with angiotensin converting enzyme 2 (ACE2)-binding sites in SARS-CoV-2 RBD in the Spike protein. CA521FALA blocks the interaction by binding all three RBDs of one SARS-CoV-2 spike trimer simultaneously. These results demonstrate the importance for antibody-based therapeutic interventions against COVID-19 and identifies CA521FALA a promising antibody that reacts with SARS-CoV-2 Spike protein to strongly neutralize its activity.

Effects of rotigotine and rotigotine extended-release microsphere therapy on myocardial ischemic injury in mice.

Rotigotine is a dopamine receptor agonist that can improve motor function in Parkinson's disease (PD) patients. Rotigotine extended-release microsphere (RoMS) is an extended-release intramuscular formulation that exhibits a sustained release of rotigotine over a 14-day period. The clinical trials of RoMS has been carried out in USA and China. The purpose of this study is to observe the effects of RoMS therapy on myocardial ischemic injury in mice, to know whether RoMS alleviate or deteriorate the myocardial ischemic injury while PD patient has onset of myocardial ischemia concurrent after administered with RoMS. A mouse model of myocardial ischemia was established using isoproterenol, and mice were pretreated with rotigotine or RoMS before inducing myocardial ischemic injury. The effects of rotigotine or RoMS therapy on the degree of myocardial ischemic injury were studied by evaluating troponin I level, creatine kinase-MB (CK-MB) activity, and histopathological changes in cardiomyocytes. The dopamine receptor blocker chlorpromazine was used to further investigate the effects of rotigotine or RoMS on myocardial ischemic injury. Furthermore, serum rotigotine concentrations were also assayed. When myocardial ischemia occurred during rotigotine or RoMS administration, troponin I level and CK-MB activity were decreased, and ischemia-induced histopathological changes in cardiomyocytes were alleviated. The effects of rotigotine were maintained only 12 h and after that no protective effect was observed. RoMS releases continuously into the circulation after intramuscular injection. The cardioprotective effects of RoMS were maintained 14 days after a single RoMS administration. When combined with chlorpromazine, the protective effects of rotigotine on myocardial ischemic injury were eliminated, and the protective effects of RoMS were also partially abolished. In the animal model of myocardial ischemia, pretreatment with rotigotine or RoMS does not deteriorate, but can alleviate cardiomyocyte injury. Furthermore, RoMS pretreatment show long-term and continuous protective effects on cardiomyocyte injury. RoMS therapy in PD patients at high risk for cardiovascular diseases may attenuate the degree of cardiomyocyte injury caused by ischemia.

LC-MS/MS method for the determination of the prodrug aripiprazole lauroxil and its three metabolites in plasma and its application to in vitro biotransformation and animal pharmacokinetic studies.

A sensitive and selective LC-MS/MS method for determination of the prodrug aripiprazole lauroxil (AL) and the three metabolites (N-hydroxymethyl aripiprazole [NHA], aripiprazole [ARP], and dehydro aripiprazole [DHA]) in plasma was developed using ARP-d8 as an internal standard. The analytes were determined on an AB Sciex Triple Quad™ 4500 system using positive ion electrospray ionization and selected multiple reaction monitoring mode. Solid phase extraction was applied for sample preparation for AL, ARP, and DHA, and protein precipitation for NHA. Chromatographic separation was performed on an Agilent Eclipse XDB-CN column (100 × 2.1 mm i.d., 3.5 µm) using the mobile phase of water and acetonitrile (25:75, v/v) containing 0.1% formic acid with a flow rate of 0.5 mL/min. The linear ranges for AL, NHA, ARP, and DHA were 0.5-50 ng/mL, 1.0-50 ng/mL, 0.5-50 ng/mL, and 0.05-5.0 ng/mL, respectively. The average recovery in the plasma sample was stable and reproducible. The precision and accuracy of the intra- and inter-run were within assay variability criteria limits. The developed method was suitable for in vitro biotransformation studies in plasma and animal pharmacokinetic studies after intramuscular injection of AL formulations.

Validated LC-MS/MS method for the simultaneous determination of rotigotine and its prodrug in rat plasma and an application to pharmacokinetics and biological conversion in vitro.

Rotigotine behenate (RGTB), a long chain alkyl ester of the prodrug of rotigotine (RGT), has been synthesized for use in a sustained delivery system. The aim of the present report was to develop and validate a simple, sensitive and reliable LC-MS/MS method for the simultaneous determination of RGT and its prodrug RGTB in rat plasma samples. Detection was performed on a 1290 Infinity UPLC coupled Triple Quad 4500 mass spectrometer operated in positive MRM mode using an Eclipse XDB-CN chromatography column (2.1mm×100mm, 3.5µm) by isocratic elution using a 0.2% formic acid aqueous solution and acetonitrile, with stable isotope labeled RGT as an internal standard. The sample preparation method employed 50µL of a plasma sample and liquid-liquid extraction with a mixture of diethyl ether-dichloromethane (3:2, v/v) as the extraction solvent. The proposed method was fully validated by assessing its specificity, linearity, precision and accuracy, recovery, matrix effects and stability. Good linearity was found within the range of 0.1-10.0ng/mL for both analytes (r>0.996). This method was successfully applied to a pharmacokinetic study of a slow release RGTB formulation in rats following a single intramuscular injection and biological conversion in vitro.